Regulation of osteoclastogenesis by three human RANKL isoforms expressed in NIH3T3 cells

Receptor activator of nuclear factor-kappaB ligand (RANKL) induces osteoclastogenesis by binding with the receptor, receptor activator of nuclear factor-kappaB in the presence of macrophage colony-stimulating factor. Three human RANKL isoforms, hRANKL1, hRANKL2, and hRANKL3, were identified. hRANKL1 was identical to previously reported RANKL and possessed intracellular, transmembrane, and extracellular domains, hRANKL2 did not have the intracellular domain, and hRANKL3 did not have the intracellular and transmembrane domains. When bone marrow macrophages were cultured with NIH3T3 cells expressing hRANKL1, osteoclasts were formed, but when cultured with NIH3T3 cells expressing hRANKL2 or hRANKL3, no tartrate resistant acid phosphatase-positive cell was observed. In the coculture system, coexpression of hRANKL3 with hRANKL1 significantly inhibited the formation of osteoclasts by hRANKL1, but coexpression of hRANKL2 with hRANKL1 did not affect the osteoclastogenesis by hRANKL1 significantly. These results suggest that the activity of osteoclastogenesis by hRANKL1 is regulated by the attenuator, hRANKL3.     

Nit1 and Fhit tumor suppressor activities are additive

The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit^?/?Nit1^?/? mice and observed that double knockout mice develop more spontaneous and carcinogen‐induced tumors than Fhit^?/? mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney‐derived double‐deficient cells appear not to activate the pChk2 pathway and when treated with H₂O₂, show little evidence of DNA damage, compared with wild type and Fhit^?/? cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas. J. Cell. Biochem. 107: 1097–1106, 2009. © 2009 Wiley‐Liss, Inc.     

Spectrocolorimetric assessment of cartilage plugs after autologous osteochondral grafting: correlations between color indices and histological findings in a rabbit model

We investigated the use of a commercial spectrocolorimeter and the application of two color models (L* a* b* colorimetric system and spectral reflectance distribution) to describe and quantify cartilage plugs in a rabbit model of osteochondral autografting. Osteochondral plugs were removed and then replaced in their original positions in Japanese white rabbits. The rabbits were sacrificed at 4 or 12 weeks after the operation and cartilage samples were assessed using a spectrocolorimeter. The samples were retrospectively divided into two groups on the basis of the histological findings (group H: hyaline cartilage, successful; group F: fibrous tissue or fibrocartilage, failure) and investigated for possible significant differences in the spectrocolorimetric analyses between the two groups. Moreover, the relationships between the spectrocolorimetric indices and the Mankin histological score were examined. In the L* a* b* colorimetric system, the L* values were significantly lower in group H than in group F (P = 0.02), whereas the a* values were significantly higher in group H than in group F (P = 0.006). Regarding the spectral reflectance distribution, the spectral reflectance percentage 470 (SRP470) values, as a coincidence index for the spectral reflectance distribution (400 to 470 nm in wavelength) of the cartilage plugs with respect to intact cartilage, were 99.8 +/- 6.7% in group H and 119.8 +/- 10.6% in group F, and the difference between these values was significant (P = 0.005). Furthermore, the a* values were significantly correlated with the histological score (P = 0.004, r = -0.76). The SRP470 values were also significantly correlated with the histological score (P = 0.01, r = 0.67). Our findings demonstrate the ability of spectrocolorimetric measurements to predict the histological findings of cartilage plugs after autologous osteochondral grafting. In particular, the a* values and SRP470 values can be used to judge the surface condition of an osteochondral plug on the basis of objective data. Therefore, spectrocolorimetry may contribute to orthopedics, rheumatology and related research in arthritis, and arthroscopic use of this method may potentially be preferable for in vivo assessment.     

Transgenic medaka enables easy oocytes detection in live fish

Easy oocyte detection in living specimens benefits various developmental biology and environmental toxicology studies. One of the earliest markers of sex differentiation in medaka (Oryzias latipes) is oocyte development. Within the field of toxicology, a simple detection method for induced oocyte in the testis (testis-ova) as a result of endocrine disruption is necessary. In this study we produced transgenic medaka whose oocytes were labeled with fluorescent proteins using the regulatory region of the 42Sp50 gene, an isoform of polypeptide elongation 1-alpha. Short (201 nt) 5'- and 3'-flanking regions were sufficient for reporter gene expression. GFP expression was first observed in female germ cells approximately 5 days post-hatching. In the mature ovaries, germ cells showed such intense fluorescence that the fluorescence was observed from outside the body wall. In contrast, very faint fluorescence was observed in the mature testes. Testis-ova, oocytes artificially induced in the testes, were also labeled with GFP. These findings indicate through the use of transgenic medaka, that detection of female germ cells was straightforward and this transgenic medaka model proves useful for tracking female germ cells in developmental and toxicological studies.     

Characterization of the crustose red alga Peyssonnelia japonica (Rhodophyta,Gigartinales) and its taxonomic relationship with Peyssonnelia boudouresquei based on morphological and molecular data

The vegetative and reproductive morphology of the crustose red alga Peyssonnelia japonica (Segawa) Yoneshigue was re‐examined based on the holotype specimen and recent collections from various localities in Japan, including the type locality, and Hawaii. This species is characterized by the following features: thallus with appressed margins, perithallial filaments arising from the entire upper surface of each hypothallial cell (the Peyssonnelia rubra‐type), easily separable perithallial filaments in a gelatinous matrix, hypothallial filaments arranged in parallel rows, unicellular rhizoids, hypobasal calcification, gonimoblasts derived mainly from connecting filaments, and spermatangia produced in a series of whorls comprised of one to four paired spermatangia surrounding each central cell (the Peyssonnelia dubyi‐type). In addition to these features, the dimensions of the vegetative and reproductive structures of Peyssonnelia boudouresquei Yoneshigue described from Brazil were consistent with those of P. japonica. Molecular phylogenetic analyses using partial 26S rDNA, rbcL, and cox2‐3 spacer DNA sequences also supported the monophyly of P. japonica (from 16 localities in Japan and one locality in Hawaii) and P. boudouresquei (from two localities in Brazil). Therefore, P. boudouresquei may be a taxonomic synonym of P. japonica. However, considering the relatively high sequence divergences between the two taxa (2.1–2.5% in partial 26S rDNA, 5.9–6.7% in rbcL, and 5.8–6.7% in cox2‐3 spacer), and the relatively limited geographic sampling ranges, we reserve the taxonomic conclusion until further morphological and genetic data of the specimens from other geographic areas connecting Japan and Brazil become available.     

Engineering of the yeast ubiquitin ligase Rsp5: isolation of a new variant that induces constitutive inactivation of the general amino acid permease Gap1

Rsp5 is an essential ubiquitin-protein ligase in Saccharomyces cerevisiae . We found previously that the Ala401Glu rsp 5 mutant is hypersensitive to various stresses that induce protein misfolding, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. To isolate new Rsp5 variants as suppressors of the A401E mutant, PCR random mutagenesis was used in the rsp5 ^A401E gene, and the mutagenized plasmid library was introduced into rsp5 ^A401E cells. As a phenotypic suppressor of rsp5 ^A401E cells, we isolated a quadruple variant (Thr357Ala/Glu401Gly/Lys764Glu/Glu767Gly) on a minimal medium containing the toxic proline analogue azetidine-2-carboxylate (AZC). Site-directed mutagenesis experiments showed that the rsp5 ^T357A/K764E cells were much more tolerant to AZC than the wild-type cells, due to the smaller amounts of intracellular AZC. However, the T357A/K764E variant Rsp5 did not reverse the hypersensitivity of rsp5 ^A401E cells to other stresses such as high growth temperature, ethanol, and freezing treatment. Interestingly, immunoblot and localization analyses indicated that the general amino acid permease Gap1, which is involved in AZC uptake, was absent on the plasma membrane and degraded in the vacuole of rsp5 ^T357A/K764E cells before the addition of ammonium ions. These results suggest that the T357A/K764E variant Rsp5 induces constitutive inactivation of Gap1.     

Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro

Background

Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases.

Methodology/Principal Findings

Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment.

Conclusions/Significance

These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.